Abstract The AIDS pandemic remains a significant global problem with over 33.3 million persons currently infected with HIV and HIV-related illnesses claiming the lives of over 1.8 million individuals annually. Various studies have suggested a link between abuse of illicit drugs, including cocaine and increased risk of HIV transmission, accelerated HIV disease progression, and AIDS-related mortality. Women represent the fastest growing population of new HIV cases. It is estimated that more than 80% of HIV cases result from sexual transmission and semen is the primary vector. However, only about 0.1% of acts of heterosexual coitus with an infected individual result in HIV infection, suggesting that factors contained in semen may modulate the infectivity or fitness of HIV particles in semen. The underlying mechanisms of HIV transmission are not well understood. In spite of the progress in studies of HIV pathogenesis and the ability of various agents to suppress virus infection, methods to prevent HIV transmission still require more investigation. Recently, we showed that semen of healthy donors contain heterologous populations of exosomes that are morphologically distinct with respect to size and electron density. These exosomes contain different species of RNA (mRNA and small RNA), and inhibit infection with HIV. Our preliminary data reveal that similar to exosomes from the semen of healthy donors, exosomes purified from HIV infected donors potently block HIV infection while exosomes from HIV infected donors who use cocaine do not inhibit HIV. Our preliminary study further showed that HIV infection and cocaine use modulates RNA composition of semen exosomes. Importantly, lysed semen exosomes from both HIV negative and HIV infected donors but not donors who used cocaine inhibit reverse transcription of lysed HIV in a cell-free system, block the synthesis of HIV U5 DNA, and reverse transcription of U5 to Gag. These data indicate direct effect of semen exosomes on HIV reverse transcription. In this application, studies are designed to 1) determine the effect of HIV infection and cocaine abuse on the anti-HIV activity of semen exosomes, 2) determine the mechanisms of anti-HIV effect of semen exosomes, and 3) characterize the effects of donor HIV status and cocaine use on semen exosome composition. As sexual transmission is the main route for the spread of HIV and semen is the primary vector, the studies proposed in this application will address a new challenge for evaluation of the efficacy of semen exosomes as a protective factor against HIV and how donor HIV status and cocaine use modulate this function.